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Fig. S2 Notum 1a inhibits Wnt/β-catenin signaling. (A-C) Notum 1a inhibits Wnt signaling in cultured cells. (A) Expression of notum1a in HEK 293 cells results in a significant, dose-dependent reduction in the relative activation of the TOPFLASH reporter to zebrafish Wnt1 (P=0.019). Co-transfection of gpc3 with notum1a potentiates the notum1a-mediated inhibition of TOPFLASH activity (P=0.013). (B) Notum1a inhibits reception of Wnt signal. Cells were transfected with wnt1 and, 4 hours post-transfection, plated with cells transfected with either topflash alone or topflash and notum1a. The reporter response of notum1a-transfected cells was significantly reduced (P=0.00002). Bars indicate standard deviations. (C) RT-PCR reveals expression of GPC1, GPC3, GPC4 and GPC5 in HEK293 cells. ACTB serves as a positive control. (D-F) Notum 1a inhibits Wnt-induced expansion of the sp5l domain. (D) sp5l at shield stage is not expressed in the animal pole of the embryo (between red bars) in wild-type embryos. (E) In wnt8-injected embryos, sp5l is ubiquitously expressed. (F) Injection of notum1a suppresses ubiquitous sp5l expression in wnt8-injected embryos (P=0.03). (G,H) Induction of notum1a during somitogenesis reduces tbx6 expression domain. (G) Heat-shocked non-transgenic embryos display normal domain of tbx6 expression at 16 somites. (H) notum1a expression at 16 somites inhibits tbx6 expression in the majority of heat shocked Tg(hs:notum1a) embryos.